Oxygen free radical and nitric oxide (NO) generation has been hypothesized to be a central mechanism of the injury that occurs on reperfusion of ischemic tissues and alterations in this process are thought to be of critical importance in myocardial preconditioning. Questions and controversy remain, however, regarding the precise role of radical generation in triggering the process of ischemic preconditioning. This knowledge is of critical importance in efforts to develop optimal strategies to protect ischemic and reperfused myocardium. Within the present grant program, EPR techniques were developed and applied to measure the process of oxygen radical and NO generation in the post ischemic heart. New instrumentation enabling in-vivo EPR spectroscopy and imaging of free radicals in whole beating hearts was also developed. In this renewal application, we propose studies to delineate the role of oxygen radical and NO generation in triggering the process of acute phase myocardial ischemic preconditioning, extending these observations from isolated buffer perfused hearts to the setting of in vivo regional ischemia as occurs with acute occlusion of a coronary artery. Direct and spin trapping EPR studies will be performed to measure, quantitate, and characterize oxygen radical and NO generation in isolated hearts and in-vivo animal models of myocardial preconditioning. The cellular mechanisms of oxygen radical and NO generation during ischemic preconditioning will be determined in order to develop optimized therapeutic approaches to induce myocardial preconditioning. Measurements of radical generation will be correlated with alterations in contractile function, high energy phosphates, and the metabolic state of the heart. Cell death will be measured by enzyme release and histology. In-vivo EPR spectroscopy and imaging will be applied to measure and spatially map myocardial oxygenation, radical metabolism and redox state during preconditioning and subsequent periods of ischemia and reflow. These experiments will provide new insight into the fundamental mechanisms that trigger ischemic preconditioning and lead to the development of effective pharmacological therapies to protect the heart.